Nikethamide adenylate



United States atent FLT/W359 Patented June 141*, 1955 NIKETHAMEDE ADENYLATE Simon L. Ruskin, New York, N. Y., assignor to Physiological Chemicals Company, New Rochelle, N. Y., a corporation of New York No Drawing. Application ()ctoher ll), 1947, Serial No. 779,209

1 Claim. (Cl. 260-4115) My invention relates to amine compounds and especially to amino acid compounds, of the individual nucleotide known as adenylic acid, and to a process for manufacturing such compounds. I

it is the general object of the present invention to provide therapeutic preparations consisting of or containing amine compounds of adenylic acid by whose use certain undesirable effects accompanyiing the use of free adenylic acid are avoided, while at the same time the therapeutic activity of certain other substances of amine character and particularly of amino acids, can be taken advantage of.

More specifically, it is an object of the invention to provide amino acid compounds of adenylic acid which are characterized by improved solubility and modified therapeutic activity, as explained more fully hereinafter.

It is a still further object of the invention to provide more or less neutral compounds of adenylic acid which are practically non-irritating on injection.

Other objects and advantages of the invention will appear as the following more detailed description of the same proceeds.

Adenylic nucleotide therapy is particularly eilective in the stabilization of the circulation, both in high and low blood pressure conditions. In the case of high blood pressure there is usually a vase-constriction with elevation of diastolic pressure. Adenylic acid has the elfect of lowering the diastolic pressure by counteracting the vaso-constriction through vase-dilatation. In the case of low blood pressure the adenylic acid, through its improvement of muscular metabolism, increases the muscular tone of the heart and improves the blood pressure level.

It is known that on the injection of certain known adenylic compounds like adenylic acid itself and sodium adenylate, there occurs a peripheral vascular dilatation accompanied by flushing of the skin and sudden drop in blood pressure which subsequently rises so that the pulse pressure is increased. However, the immediate vascular reaction is in some cases sufiicient to cause syncopy and accompanying dilatation of the coronary artery, while increase of the flow of blood to the heart muscles gives the patient Who has a labile vasomotor mechanism a sense of tightness in the chest which may be distressing, despite the fact that the patient is in no real danger. With rising blood pressure this feeling disappears. This undesirable reaction has militated against a wider use of the adenylic nucleotide.

I have found that certain of the amine compounds of adenylic acid counteract these disturbances by slowing the speed of utilization of the adenylic nucleotide, thereby diminishing the etfect of the disturbing factors, and thus provide a less intense but longer-lasting adenylic effect. By combination with an amine, the adenylic acid is made much more suitable for injection not only because of the decrease in acidity but generally also because of the improvement in the solubility. Thus, the monoeth-anolamiue salt of adenylic acid has a pH close to neutral, is much more soluble in water than adenylic acid, and is non-irritating on injection.

The amine component of the amine compounds of adenylic acid of the present invention may or may not in itself have therapeutic activity. Thus, in the case of the mono-, di-, and tri-ethanolamine, propanolamine and similar non-toxic alkylolamine salts, and the pyridine salt or adenylic acid, the amine base acts to reduce the rate of the adenylic acid eifect without adding any particular therapeutic activity to the compound, there resulting a sustained and prolonged physiologic adenylic action. On the other hand, amino acids, like those obtained by hydrolysis of proteins, vitamin B- components, and other amine compounds referred to elsewhere herein, contribute their own specific effects while at the same time moderating the action of the adenylic nucleotide.

The amines whose adenylic acid compounds are included within the scope of the present invention are of various types. Important physiological ellects are obtained when the adenylic acid is combined with various amino acids, and particularly with arginine, histidine, lysine, tryptophane, gylcine, valine, cystine and cysteine. These compounds enhance the energy potential of the adenylic nucleotide and apparently facilitate the use of the energy-rich iminoalkylol phosphate bond which is present in adenylic acid. Another group of amines which form therapeutically desirable compounds with adenylic acid are creatin, creatinine, creatinine-urea, alkaloid hormones containing amino groups, urea, glutathione, and the amino compounds, as a group, contained in liver extract. The invention contemplates the production also of adenylates of other amino compounds like the protamines, glucosamine, succinarnide and pyridine.

In accordance with the invention, therefore, adenylic acid is combined with amines of such character that the normal adenylic action is modified or retarded; or the physiological action of the amine, where the same has physiological activity, is modified.

The dosage of the amine adenylates embraced by the present invention can correspond to that of adenylic acid or of the amine, whichever is the lower. however, the dosage of the adenylate radical, or of the physiologically active amine radical, can be somewhat lower than the normal dosage for such radical when administered without the other, where the action of the two radicals is similar; where, however, they act antagonistically somewhat larger dosages can be safely administered.

In preparing the compounds of the present invention, the amine can be treated with adenylic acid in combining proportions in aqueous solution, preferably with the aid of heat. The amine adenylate canbe recovered by evaporation or by precipitation by a water-miscible organic liquid in which the adenylate is relatively insoluble, like lower aliphatic alcohols, acetone, etc. Where the amine is employed in the form of its acid salt, a neutralizing agent like an alkali metal base can be added to bind the acid. Where the amine chloride is used, the chlorine can be bound also by silver.

The invention will be described in further detail by way of the following examples which are presented for purposes of illustration and not as indicating the scope of the invention.

Example 1.Gluc0samine adenylate 120 g. glucosaminhydrochloride were dissolved in 2000 cc. water, and a solution of 80 g. silver carbonate in 1500 cc. water containing 40 cc. concentrated sulfuric acid was added. Glucosaminsulfate was formed and the solution was kept acid so as to avoid reduction of silver salt and oxidation of the glucosamin. The mixture was stirred and shaken, allowed to stand an hour in the dark, then filtered through a Biichner filter. The filterat'e was cloudy In general,

with some colloidal silver chloride and was heated to near boiling to precipitate the silver chloride. After standing in the dark overnight, it was filtered on an uncoated Biichner filter. The small precipitate was not washed. The filtrate was clear and a few drips of it gave a negative test for chloride with silver nitrate.

Hydrogen sulfide was passed through the filtrate to precipitate out traces of silver and the silver sulfide filtered off. To the hot filtrate was added a little less than 220 g. of Ba(OH)2.8H2O in 1000 cc. hot water and all residual sulfate ions removed by further quantities of the base until the exact amount of barium hydroxide was added. as shown by testing small portions of filtrate with a drop of N/H2SO4. (The use of excess of barium hydroxide is to be avoided as it may affect the glucosamine.) Test samples may be combined with the main portion to avoid loss.

The filtrate fromthe barium sulfate was evaporated in vacuo and made up to a volume of 4800 cc. with water.

To 1640 cc. of this glucosamin solution, equivalent to 40.96 g. glucosamine hydrochloride, were added 66 g. adenylic acid were dissolved in 1000 cc. hot water, and the mixture cooled, filtered through a carbon-covered filter, evaporated in vacuo to about 150 cc., poured into a small beaker, the flask washed with 50 cc. warm water and this combined with the main portion. Ethyl alcohol was stirred in until permanent turbidity was obtained, and the mixture was then placed in a desiccator. (It is better to dissolve the adenylic acid in water before adding to the glucosaminc solution in order to avoid warming.)

On evaporation, hair-like crystals appeared on the sides of the beaker, but the main portion was a hard mass.

The product has a tendency to darken; heating of the solution above 50 C. should therefore be avoided.

Alkylated glucosamines, like methyl glucosamine, can be used instead of glucosamine to produce the corresponding alkyl glucosarn-ine adenylate.

Example 2.-Histldine adcrzylate To 10 grams of adenylic acid in 100 cc. water there were added 44 cc. of a 5% histidine solution equivalent to 2.2 histidine. Solution of the adenylic acid was slow but when heated to about 60 C. it dissolved rapidly. The solution was filtered through a carbon-coated filter to clear it of a slight turbidity. A little ethyl alcohol was added until a slight permanent turbidity was obtained. A crystalline precipitate formed over a period of several days. Addition of more alcohol increased the yield of crystals of histidine adenylate.

Example 3.Mn0ethan0la1nine adenylate Ten grams of monoethanolamine were dissolved in water and made up to a volume of 500 cc. with water. To 88 cc. of this solution, equivalent to 1.76 g. monoethanolamine, were added 10 grams of adenylic acid. Most of the adenylic acid went into solution. A little ethyl alcohol was added until a slight turbidity was obtained. After several days the solution was filtered from the small residue. To the filtrate there was added a little butyl alcohol, and the mixture allowed to stand in the desiccator to evaporate. Hair-like crystals formed on the sides of the beaker. The product appeared to be slightly deliquescent.

Example 4.Clt0line adenylate 1500 g. choline chloride were dissolved in 6000 cc. methyl alcohol to which were then added 2750 g. silver carbonate. The mixture was stirred well, the silver carbonate at bottom of solution being stirred up, and then allowed to stand for several hours in the dark. The mixture was filtered through a carbon-coated filter, and the residue washed with methyl alcohol. The total volume of choline carbonate solution was 8650 cc.

200 cc. methyl alcohol were added to 10 g. adenylic acid. To the mixture there were added 23 cc. of the choline carbonate solution as prepared above, equivalent to about 4.0 g. choline chloride. Since the adenylic did not all go into solution, a little water was added and the solution heated on the hot water bath. The product was iltered through a carbon-coated filter and the filtrate placed in the desicccator to evaporate. The hair-like crystals which formed on the sides of the beaker were very deliquescent.

Example .H istamine adenylate To a solution of 111 mg. of histamine (M/l000) in a few cc. of water, there were added 694 mg. of adenylic acid (M/500). The mixture was shaken until clear and then made up to 16 cc. with water, thereby yielding a 5% solution of histamine adenylate.

Example 6.Glutathi0ne adenylate 0.35 g. of adenylic acid (M/lOOO) were dissolved in 20 cc. of hot water. To this there were added 0.30 g. of glutathione (M/ 1000). The clear reaction solution was then concentrated in vacuo. On standing for some time in the ice chest the solution yielded white crystals of the glutathione adenylate.

Example 7.Acetylch0line adenylate 0.6 g. of adenylic acid were dissolved in cc. of water to which had been added 1.5 cc. of normal NaOH solution. To this there was added 0.3 g. of acetylcholine hydrochloride dissolved in about 2 cc. of water. The resulting solution was still acid. A few drops of normal sodium hydroxide solution were added to bring the solution to neutral. The final volume of the solution is about cc. It was unnecessary to remove the formed sodium chloride as its presence is unobjectionable in a solution prepared for injection.

Example 8.Nikellzamide adenylal-e 1.95 g. of adenylic acid were partially dissolved in 100 cc. of warm water and 1 g. of nil-:ethamide added to the mixture. The latter was stirred and the two reagents dissolved almost completely. The solution was filtered with suction from a small residue. The filtrate was evaporated in vacuo to about 10 cc., when crystals began to form. The syrup was transferred to a small beaker and placed in the desiccator. After standing for three days the crystals were filtered free of the mother liquor.

Example 9.Pyridine adenylate 2 grams of adenylic acid and 0.51 cc. of pyridine (equimolecular proportions) were added to cc. of hot water in which both reagents dissolved almost completely. The solution was filtered hot through filter paper. The filtrate was clear. On cooling, methyl alcohol was added to the filtrate until a permanent turbidity was obtained. The solution was placed in a desiccator and on further evaporation therein, crystals of the salt were obtained. The addition of propyl alcohol to the thin syrup resulted in better crystals. The crystals were filtered and washed with two portions of 10 cc. of propyl alcohol.

Example 10.Cystine adenylute 2 grams of adenylic acid (2 rnols) and 0.7 g. of cystine (1 mol) were heated in cc. of water to about C. The solution was filtered hot from an insoluble residue which weighed 0.8 g. Crystals formed in the filtrate on cooling. After several days the crystals of cystine adenylate were filtered and dried. Weight of crystals: 0.9 g.

Example ]1.Trypt0pl1ane allenylate 2 grams of adenylic acid (1 mol) and 1.2 grams of tryptophane (1 mol) were heated together in about 50 cc. of water to about C. The reagent is dissolved almost completely. The solution was filtered hot from the very small residue. On cooling, crystals of the salt formed and were filtered and dried. Weight of crystals: 2.4 g.

Example 1Z.Urea adenylate 1 gram of adenylic acid (1 mol) and 0.17 gram of urea (1 mol) were heated in about 50 cc. of water to about 60 C. The reagents dissolved completely for a residue which weighed 0.2 g. The solution was filtered and the filtrate evaporated to about 15 cc. Crystals of the salt formed and after several days were filtered and dried. Weight of crystals: 0.6 g.

Example 13.Pr0mmine adenylate 0.5 g. of protamine sulfate was freed from sulfate radical by precipitation of the latter with barium hydroxide solution (10 per cent). About 20 per cent of the protamine sulfate was found to be actually sulfate. To the filtrate, there was added 0.85 g. of adenylic acid partially dissolved in 50 cc. of hot water. On slight heating with the protamine filtrate the suspended adenylic acid went into solution. The solution was concentrated in vacuo to a thin syrup of about 10 cc. To a part of this syrup there was added 20 cc. of absolute ethyl alcohol and the mixture evaporated in a desiccator over calcium chloride. There was obtained a dried, glistening white residue which appeared to be crystalline.

Example 14.Lysine adenylate 350 mg. adenylic acid and 200 mg. lysine hydrochloride were suspended in 44 cc. water. On addition of 1 cc. normal sodium hydroxide, complete solution took place at room temperature. After vacuum desiccation a white amorphous powder was obtained.

Example 15.-Arginine adenylate 35 mg. adenylic acid and 210 mg. arginine hydrochloride were suspended in 4 cc. water. On addition of 1 cc. normal sodium hydroxide, a colorless solution resulted which was placed in a vacuum desiccator. On complete evaporation a white amorphous powder was obtained.

In similar fashion the adenylates of the other amine compounds referred to above can be prepared. As indicated by certain of the examples, where the amine is used in the form of an acid salt, like the hydrochloride, enough of a non-toxic base, like sodium, potassium or calcium hydroxides or carbonates should be added to neutralize the acid.

The above compounds may be administered by mouth but in general are preferably administered by injection intramuscularly. I have found that where it is desirable to delay the rate of absorption of the adenylic radical, the adenylates are advantageously suspended in a mixture of beeswax and peanut oil. Suspensions in these media are especially desirable for patients who are sensitive to the circulatory influence of the adenylates. In place of the mixture of beeswax and oil, other oils and watermiscible, non-toxic organic solvents can be used, and likewise other waxy substances, such as a mixture of propylene glycol and cholesterol, or a mixture of peanut or sesame with cholesterol. These suspending agents can be used advantageously also for sodium and potassium adenylates whether used alone or in admixture with any of the amine adenylates above disclosed. Where an increased rate of absorption of the adenylic radical is desired, suspensions of creatin adenylate and creatinine adenylate in an aqueous medium have proved highly effective. These compounds can be prepared as above by heating combining proportions of adenylic acid and creatin or creatinine in an aqueous solvent, followed by evaporation.

I claim:

Nikethamide adenylate.

Degering: Outline of Organic Nitrogen Compounds, 1945, page 615-616 (2 pp.). 

1. NIKETHAMIDE ADENYLATE. 